Enhancement Of Radiotherapeutic Efficacy For Esophageal Cancer By Paclitaxel-loaded Red Blood Cell Membrane Nanoparticles Modified By The Recombinant Protein Anti-EGFR-iRGD

Chemoradiotherapy plays an important role in comprehensive treatment for esophageal cancer?EC?.The prognosis for esophageal cancer after chemoradiotherapy remains dismal because of markedly high locoregional recurrence and distant metastasis rate.How to improve the efficacy of chemoradiotherapy in esophageal cancer becomes the target urgently needed to resolve.Multiple clinical trials have shown that blindly increasing the radiation dose to EC does not improve efficacy but may increase radiation damage to normal tissues.Therefore,it becomes one of the most attractive highlights that how to improve the efficacy and radiosensitizing effect of chemotherapy agents for patients treated with concurrent chemoradiotherapy.Considering that the most common radiosensitizers in current clinical applications are still chemotherapeutics,the development of new intelligent medications to promote synergistic effects with radiation is a promising research direction.However,conventional chemotherapeutic drugs have some common limitations,such as a lack of tumor targeting,poor tumor tissue penetration,and the absence of a sustained-release effect.Developing a nanoparticle?NP?-based drug delivery system with unique properties is an essential means of rapidly overcoming the above limitations of chemotherapeutic agents.In the first part of the study,on the basis of the construction of anti-EGFR-iRGD,a fusion protein with dual tumor targeting and high penetration properties,our research team had established a PTX-loaded red blood cell?RBC?membrane NPs?PRNPs?drug delivery system with water solubility and sustained drug release effect?PRNPs?.The recombinant protein anti-EGFR-iRGD was further designed to conjugate to the surface of PRNPs,thereby forming a new drug-loaded nanoparticle with dual targeting,high tumor penetration and sustained release properties?iE-PRNPs?.In the second part,we studied the radiosensitizing effect and mechanism of the novel nanoparticle iE-PRNPs on esophageal cancer cell lines with high expression of EGFR,and identified its value as a potential effective radiosensitizer for esophageal cancer,providing a basis for improving the level of comprehensive treatment of esophageal cancer.Part?Construction and verification of paclitaxel-loaded red blood cell membrane nanoparticles modified with fusion proteinanti-EGFR-iRGDObjective:To construct the paclitaxel-loaded red blood cell membrane nanoparticles modified with anti-EGFR-iRGD,characterize their physical properties,and verify their sustained release,targeting and tumor penetration ability.Methods:Firstly,RBC membrane ghosts were first prepared by hemolysis in hypotonic medium.After ultrasonic treatment in the ice bath,the PTX ethanol solution was rapidly added to form a nanoparticle mixture,and the unloaded PTX crystals and large particles were removed by low speed centrifugation.The obtained supernatant was again subjected to high-speed centrifugation and redispersed in PBS to generate PTX-loaded red blood cell membrane nanoparticles?PRNPs?.Then,PRNPs was maleimide-functionalized by sulfo-smcc through the reaction between the NHS-ester moiety and the primary amine group of the cell membrane protein.Simultaneously,the thiolation of anti-EGFR-iRGD was conducted with Traut's reagent.Finally,after removing the byproducts by ultrafiltration,the thiolated anti-EGFR-iRGD was incubated with the maleimide-functionalized PRNPs to generate the iE-PRNPs.After preparation,iE-PRNPs were characterized by fluorescence microscopy,dynamic light scattering?DLS?method,transmission electron microscopy?TEM?,and X-ray photoelectron spectroscopy?XPS?.The drug loading efficiency?LE?,loading content?LC?and PTX release in vitro of PRNPs and iE-PRNPs were detected by high-performance liquid chromatography?HPLC?.Western blotting was used to identify esophageal cancer cell lines with different EGFR expression levels.Fluorescence microscopy and flow cytometry were used to observe the uptake of iE-PRNPs in esophageal cancer cell lines with different EGFR expression levels.Three-dimensional multicellular spheres?3D MCSs?were cultured using esophageal cancer cell KYSE-450 with high expression of EGFR.The penetration of iE-PRNPs into 3D MCSs was determined by confocal laser scanning microscopy?CLSM?.Results:1.After the successful preparation of PRNPs nanoparticles,the drug LE and LC values were 56.1%and 22.4%,which were determined by HPLC.After anti-EGFR-iRGD conjugation,the iE-PRNPs showed an increase in the DLS measured diameter from 147.6±1.4 nm to 171.5±4.6 nm,with a relatively low polydispersity?0.319±0.022?and a significantly reduced zeta potential from-23.6±0.2 mV to-28.2±0.2 mV.XPS was employed to identify a change in the nitrogen signal of the iE-PRNP surface chemistry.A characteristic peak enhancement at 398 eV?signals from nitrogen?was apparent in the XPS spectrum of the iE-PRNPs,demonstrating the successful modification of the PRNPs with anti-EGFR-iRGD.Under the TEM,it could be seen that the iE-PRNPs particles were round and uniform in size and the diameter was about 170nm.2.The PTX release in vitro was measured by HPLC.The release of PTX from PRNPs was found to be time-dependent,with a 41.25±0.33%drug release in the first24 h and an approximately 60.53±0.56%release within one week.Additionally,no significant difference in PTX release was observed when the PRNPs were modified with anti-EGFR-iRGD to generate iE-PRNPs.3.EGFR expression levels in human EC cell lines were first investigated by a Western blotting assay.The result indicated that EGFR is overexpressed in KYSE-450 cells and has low expression in TE-1 cells.The binding affinity experiments confirmed that the affinity of anti-EGFR-iRGD to KYSE-450 with high expression of EGFR is higher than that of TE-1 with low expression of EGFR.4.The cellular uptake of iE-PRNPs was observed using FM and FCM.KYSE-450 cells treated with iE-PRNPs presented higher DiO fluorescence intensity in the cytoplasm than cells treated with PRNPs.Furthermore,the difference in cellular uptake was quantitatively investigated by FCM analysis.The mean fluorescence intensity?MFI?represents the number of NPs taken up by the cells.The uptake of iE-PRNPs was increased by 2.57-fold compared with that of PRNPs,which showed that anti-EGFR-iRGD modification could enhance the tumor targeting of PRNPs.5.KYSE-450 cells were cultured into 3D MCSs and used to investigate the penetration and accumulation profiles of iE-PRNPs.MCSs were cultured separately with DiO-labeled PRNPs and iE-PRNPs.The penetration of PRNPs mostly appeared in the peripheral cells of the MCSs.Only weak fluorescence from PRNPs was observed in the middle of the MCSs,indicating that only a small number of PRNPs can penetrate through the spheroids.In contrast,for iE-PRNPs,the DiO fluorescence signal appeared not only in the periphery of the MCSs but also throughout nearly the entire MCSs.This result indicated that anti-EGFR-iRGD modification could enhance the penetration of PRNPs,resulting in more efficient delivery of PTX into tumor cells.Conclusions:In the first part of the study,PTX-loaded RBC membrane nanoparticles?PRNPs?were successfully prepared,and the fusion protein anti-EGFR-iRGD was further modified onto the surface of PRNPs nanoparticles to successfully generate novel nanoparticles,namely iE-PRNPs.Characterization studies show that iE-PRNPs are uniform in size,well dispersed,and have a sustained release effect.It was further confirmed that iE-PRNPs possessed tumor-targeting,high penetrability,and sustained-release properties that free PTX does not possess.In conclusion,iE-PRNPs integrates the dual targeting and high tumor penetration properties of the fusion protein anti-EGFR-iRGD,as well as the sustained release properties of PRNPs,which appear to be an ideal new nano-drug.Part?Enhancement of radiotherapeutic efficacy andmechanism research for esophageal cancer by paclitaxel-loadedred blood cell membrane nanoparticles modified by therecombinant protein anti-EGFR-iRGDObjective:To investigate the radiosensitizing effect and mechanism of the novel paclitaxel-loaded red blood cell membrane nanoparticles modified by the recombinant protein anti-EGFR-iRGD,iE-PRNPs,on esophageal cancer cell lines with high expression of EGFR.Methods:Firstly,the cytotoxic effects of iE-PRNPs and PRNPs on KYSE-450 and TE-1were evaluated in vitro using MTT assay,and the corresponding IC50 and IC15concentrations were obtained.The following radiosensitization experiments were performed using the IC₁₅ concentrations of PTX,PRNPs,and iE-PRNPs to ensure that the radiation-enhancing effect was not due to direct cytotoxicity.Secondly,the clonogenic survival curves of cells after different combinations of treatments and a gradient of RT doses were obtained using clonogenic survival assay.The SERs of free PTX,PRNPs,and iE-PRNPs were calculated.To analyze the difference of radiotherapeutic radiosensitizing effect in esophageal cancer cell lines with different EGFR expression levels of iE-PRNPs and PRNPs.Finally,KYSE-450 esophageal cancer cell lines with high expression of EGFR were treated with PTX,PRNPs and iE-PRNPs,respectively.Flow cytometry was used to assess the difference in radiosensitive G2/M phase arrest.The KYSE-450 EC cells with high expression of EGFR were treated with PTX,PRNPs and iE-PRNPs,followed by IR at a dose of 6Gy.DCFH-DA was used as a probe to detect ROS generation in the cells after IR.The FCM was used to measure the MFI differences between groups to assess the number of reactive oxygen species?ROS?produced after IR.?-H2AX immunofluorescence staining was performed to evaluate DNA DSBs in the cells after treatment.Similarly,after staining with?-H2AX immunofluorescence staining,fluorescence microscopy was used to assess the difference in DNA DSBs produced by different groups.Results:1.In anti-tumor studies in vitro,PTX,PRNPs and iE-PRNPs exhibited significant cell cytotoxicity against the KYSE-450 and TE-1 cell lines.The inhibition rates were dependent on the PTX concentration and incubation time.The iE-PRNPs group exhibited 0.82-fold lower IC50 values than the PRNPs group in the KYSE-450 cell line after 72 h of treatment.Similarly,the iE-PRNPs group showed 0.9-fold lower IC50 values than the PRNPs group in the TE-1 cell line after 72 h of treatment.The cytotoxic enhancement effect of iE-PRNPs that was seen in the TE-1 cell line even with a low expression of EGFR may be attributed to the dual tumor-targeting strategy and high tumor penetration of the fusion protein anti-EGFR-iRGD.The IC15 values of PTX,PRNPs and iE-PRNPs were calculated by the linear regression method for later use.2.A clonogenic survival assay was carried out to evaluate the in vitro radiosensitizing effect of iE-PRNPs.The sensitizer enhancement ratios?SERs?of PTX,PRNPs,and iE-PRNPs were calculated.Compared with those of PTX,the SERs of PRNPs and iE-PRNPs increased by 1.04-fold and 1.32-fold,respectively,in the KYSE-450 cells and 1.18-fold and 1.25-fold,respectively,in the TE-1 cells.These results revealed that the nanoparticular formulation could improve the radiosensitization of free PTX.Specifically,the SER value of iE-PRNPs in the KYSE-450 cell line was 1.51,while that of PRNPs was 1.19,which represents a1.27-fold difference.Comparatively,the SERs were 1.97 for iE-PRNPs and 1.86 for PRNPs in the TE-1 cell line,which represents a 1.06-fold difference.The above results indicated that compared with PRNPs,iE-PRNPs had a more significant advantage in radiosensitization in the KYSE-450 cell line with high expression of EGFR,whereas in the TE-1 cell line with low EGFR expression,the difference was slight.3.FCM analysis of cell cycle distribution showed that after treatment of KYSE-450 cells with PTX,PRNPs and iE-PRNPs,the proportion of cells arrested in G2/M phase were 10.21%,12.24%,and 14.96%,respectively.The treatment of KYSE-450 cells with iE-PRNPs further increased the proportion of cells arrested in the G2/M phase,with values 1.22-fold,1.47-fold and 1.80-fold higher than those in the PRNPs,PTX and control groups,respectively.These results demonstrated that iE-PRNPs blocked more radiosensitive G2/M phase cells than both PRNP and PTX during IR exposure,which was beneficial for improving radiosensitizing efficacy.4.DCFH-DA was used as a probe to detect ROS generation in the cells after IR.The MFI detected by FCM represents the intracellular ROS level.The MFIs of the IR alone,PTX+IR,PRNPs+IR,and iE-PRNPs+IR groups were increased by 2.03-fold,2.39-fold,9.16-fold,10.86-fold compared to that of the control,respectively.Compared with those of the PTX+IR and PRNPs+IR groups,the MFI of the iE-PRNPs+IR group increased by 4.54-fold and 1.19-fold,respectively,demonstrating that the iE-PRNPs could induce more ROS formation than PTX or unmodified PRNPs when combined with IR.5.DNA DSBs in KYSE-450 cells were investigated using?-H2AX immunofluorescence staining.Only a negligible?-H2AX signal was observed in KYSE-450 cells treated with IR alone,and a low level of?-H2AX fluorescence was exhibited in the cells treated with PTX or PRNPs after IR exposure.In contrast,a substantially higher level of?-H2AX fluorescence was observed within the nuclei of cells in the iE-PRNP+IR group.The above data demonstrated the presence of increased DNA DSBs in response to IR after treatment with iE-PRNPs,which is associated with radiosensitizing effect of iE-PRNPs.Conclusions:In the second part of the study,in vitro anti-tumor studies showed that PRNPs and iE-PRNPs had significant cytotoxic effects on both KYSE-450 and TE-1.Compared with PRNPs,iE-PRNPs showed more enhanced the cytotoxicity of esophageal cancer cells with different EGFR expression levels,which was related to the bispecific tumor targeting and high tumor penetration performance of the fusion protein anti-EGFR-iRGD.Clonogenic survival assays showed that PTX-loaded nanoparticles could enhance the radiosensitization of free PTX.Compared with PRNPs,iE-PRNPs had more obvious radiosensitization advantages in KYSE-450 cell lines with high EGFR expression.Mechanism studies revealed that the improved radiosensitization efficacy was associated with enhanced G2/M arrest,increased ROS,and more effective DSB induction.These findings suggested that iE-PRNPs could serve as a potential radiosensitizer for EC.